MYL9 Antibody, Biotin conjugated

What is MYL9 and why is it important in cellular research?

MYL9 (Myosin Light Chain 9) functions as a myosin regulatory subunit that plays a critical role in regulating both smooth muscle and nonmuscle cell contractile activity through phosphorylation mechanisms. Its importance stems from its involvement in fundamental cellular processes including cytokinesis, receptor capping, and cell locomotion . Recent research has revealed that in myoblasts, MYL9 may regulate PIEZO1-dependent cortical actomyosin assembly involved in myotube formation .

The protein is also known by several alternative names including MLC2, MRLC1, MYRL2, 20 kDa myosin light chain, MLC-2C, Myosin RLC, and Myosin regulatory light chain MRLC1 . Understanding MYL9 function is particularly important for researchers investigating cellular motility, muscle contraction mechanisms, and related pathological conditions.

What applications are biotin-conjugated MYL9 antibodies most commonly used for?

Biotin-conjugated MYL9 antibodies are primarily employed in the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Recommended dilution typically 1:1000

• Immunohistochemistry (IHC): Recommended dilution ranges of 1:10-50 or 1:50-100 depending on specific antibody preparations

• Western Blotting: Typically used at dilutions between 1:100-500 up to 1:1000

• Immunofluorescence/Immunocytochemistry: Used for cellular localization studies

These conjugated antibodies are particularly valuable in detection systems that leverage the strong biotin-avidin/streptavidin interaction, allowing for signal amplification in various immunodetection techniques. The conjugation does not significantly alter the biological activity of the antibody due to biotin's relatively small molecular size .

How do I properly validate a biotin-conjugated MYL9 antibody for my specific application?

Validating a biotin-conjugated MYL9 antibody requires a systematic approach:

• Positive and negative controls:

  • Use tissues/cells known to express MYL9 (e.g., smooth muscle tissue from colon as shown in validated IHC applications)

  • Include a negative control without primary antibody and samples known not to express MYL9

• Specificity testing:

  • Perform peptide competition assays using the immunogen (typically synthetic peptides within Human MYL9 aa 1-50)

  • Test cross-reactivity with related proteins (particularly MYL12A, MYL12B)

  • For phospho-specific antibodies, compare results with and without phosphatase treatment of samples

• Application-specific validation:

  • For IHC: Optimize fixation conditions (formalin-fixed, paraffin-embedded tissues are commonly used)

  • For Western blot: Confirm expected molecular weight (approximately 20 kDa for MYL9)

  • For dot blot analysis: Test with phosphorylated and non-phosphorylated peptides

• Document specific reactivity patterns:

  • In Western blots, MYL9 typically appears as a band of approximately 18-20 kDa

  • For phospho-specific antibodies, verify increased signal after treatments known to induce phosphorylation (e.g., calyculin treatment)

What are the optimal storage conditions for maintaining biotin-conjugated MYL9 antibody activity?

Maintaining optimal biotin-conjugated MYL9 antibody activity requires careful handling:

• Temperature considerations:

  • Store unopened antibodies at 2-8°C for up to 1 year

  • For opened/reconstituted products, maintain at 2-8°C for up to 1 month

  • Avoid repeated freeze-thaw cycles

• Solution parameters:

  • Most preparations are stored in phosphate-buffered saline (PBS)

  • Many contain preservatives such as 0.09% sodium azide

  • Typical protein concentration is approximately 1.0 mg/ml

• Avoiding contamination:

  • Use sterile technique when handling

  • Aliquot into smaller volumes if frequent usage is anticipated

  • Note: For some antibody preparations, aliquoting is not recommended

• Reconstitution guidance:

  • For lyophilized standards or antibodies, follow manufacturer reconstitution protocols

  • Not recommended to reuse after redissolving

What are the critical steps in developing a reliable conjugation protocol for creating biotin-labeled MYL9 antibodies?

Developing a reliable conjugation protocol for biotin-labeling MYL9 antibodies involves several critical steps:

• Antibody preparation:

  • Ensure antibody purity (consider using protein A/G purification if necessary)

  • Adjust antibody concentration to optimal range (typically 1-10 mg/ml)

  • Remove any buffer components containing primary amines or sulfhydryl groups

• Selection of appropriate biotinylation reagent:

  • NHS-activated biotin esters for primary amine conjugation

  • Maleimide-activated biotin for sulfhydryl coupling

  • Choose water-soluble vs. non-water-soluble reagents based on application needs

• Optimization of reaction conditions:

  • Maintain pH between 7.0-9.0 for amine-reactive biotinylation

  • Control molar ratio of biotin:antibody (typically 5:1 to 20:1)

  • Monitor reaction time (typically 30 minutes to 2 hours at room temperature)

• Purification of conjugate:

  • Remove free biotin using gel filtration chromatography

  • Consider using metal chelate affinity chromatography for purification

  • Monitor elution with appropriate methods (typically UV absorbance)

• Validation of biotinylation efficiency:

  • Calculate the biotin:protein ratio using colorimetric assays

  • Assess retained antibody activity through functional assays

  • Compare activity to non-conjugated control antibody

How can phospho-specific MYL9 antibodies be used to investigate signaling pathways in cancer progression?

Phospho-specific MYL9 antibodies offer powerful tools for investigating signaling pathways in cancer:

• Detecting activated MYL9 in cancer tissues:

  • Phosphorylation at Ser19 and Ser20 is critical for MYL9 activation

  • Recent studies show MYL9 upregulation in squamous cervical cancer (SCC) tissues compared to peritumoral samples

  • Expression levels correlate with cancer stage as shown in the following data from patient samples:

  Patient	Stage	MYL9 mRNA expression
  5	IB1	14.510 ± 1.0660
  7	IIA2	19.180 ± 0.4058
  19	IIA2	10.080 ± 0.9134
  25	IB2	8.6230 ± 0.2416
  30	IB2	0.2448 ± 0.0638

• Pathway analysis using phospho-specific antibodies:

  • MYL9 promotes JAK2/STAT3 pathway activity in cancer cells

  • Knockdown of MYL9 decreases phosphorylation of JAK2 and STAT3

  • Connected to aerobic glycolysis, with effects on GLUT1, HK2, and LDHA expression

• Experimental approaches:

  • Western blotting with phospho-specific antibodies before and after pathway inhibitor treatment

  • Immunohistochemistry to map phospho-MYL9 distribution in tumor tissue sections

  • Correlation of phospho-MYL9 levels with metastatic potential in experimental models

• Translational significance:

  • MYL9 can function as either tumor suppressor or oncogene depending on cancer type

  • In cervical cancer, MYL9 promotes migration and invasion, making it a potential biomarker for targeted treatment

What strategies can improve signal specificity when using biotin-conjugated MYL9 antibodies in multiplex imaging systems?

Improving signal specificity in multiplex imaging with biotin-conjugated MYL9 antibodies requires sophisticated approaches:

• Blocking endogenous biotin:

  • Pretreat tissues with avidin/streptavidin followed by free biotin

  • Use commercial endogenous biotin blocking kits

  • Employ alternative detection systems for tissues with high endogenous biotin

• Sequential detection strategies:

  • Apply stripping protocols between detection cycles

  • Use tyramide signal amplification (TSA) with different fluorophores

  • Consider microwave-based antibody elution between cycles

• Advanced multiplexing approaches:

  • Mass cytometry (CyTOF) using metal-tagged secondary reagents

  • Conjugation-ready formats designed specifically for fluorochromes, metal isotopes, oligonucleotides, and enzymes

  • Employ cyclic immunofluorescence (CycIF) protocols

• Image analysis optimization:

  • Implement spectral unmixing algorithms

  • Use computational approaches to reduce autofluorescence

  • Apply machine learning for signal identification and separation

• Controls for signal validation:

  • Include single-stained controls

  • Use isotype controls conjugated with biotin

  • Perform antibody titration to determine optimal concentration

How can I effectively use biotin-conjugated MYL9 antibodies to study the role of MYL9 phosphorylation in cell motility and contraction mechanisms?

Studying MYL9 phosphorylation in cell motility and contraction requires sophisticated approaches:

• Live-cell imaging systems:

  • Combine biotin-conjugated phospho-specific MYL9 antibodies with cell-permeable streptavidin-fluorophore conjugates

  • Use microinjection of labeled antibodies for real-time tracking

  • Consider FRET-based approaches to detect phosphorylation events

• Correlation with functional assays:

  • Transwell and Boyden assays have demonstrated that MYL9 knockdown significantly suppresses cancer cell migration and invasion

  • Combine migration assays with phospho-MYL9 staining patterns

  • Use traction force microscopy to correlate phospho-MYL9 levels with mechanical forces

• Phosphorylation site-specific studies:

  • MYL9 can be phosphorylated at threonine 18 and serine 19/20 positions

  • Different phosphorylation patterns generate distinct functional outcomes

  • Use site-specific phospho-antibodies to distinguish these patterns:

    • Anti-MYL9 (pS20) + MYL12A (pS19) + MYL12B (pS20) antibodies

    • Phospho-MYL9 (Ser19) antibodies

• Experimental manipulations:

  • Use calyculin treatment (100 ng/ml for 15-30 minutes) to induce phosphorylation

  • Employ phosphatase inhibitors to preserve phosphorylation state

  • Validate with alkaline phosphatase treatment as negative control

What are the major sources of background in biotin-conjugated antibody detection systems, and how can they be minimized?

Major background sources in biotin-conjugated antibody systems include:

• Endogenous biotin interference:

  • Particularly problematic in biotin-rich tissues (liver, kidney, brain)

  • Solution: Block endogenous biotin using avidin/biotin blocking kits

  • Alternative: Heat pretreatment (microwave or pressure cooker) can denature endogenous biotin

• Non-specific binding of detection reagents:

  • Streptavidin/avidin can bind non-specifically to charged molecules

  • Solution: Include additional blocking proteins (BSA, casein, or commercial blockers)

  • Optimize salt concentration in wash buffers (150-300 mM NaCl)

• Excessive antibody concentration:

  • Over-concentrated antibody increases background signal

  • Solution: Perform antibody titration experiments (typical starting dilutions for MYL9 antibodies range from 1:100-1:1000)

  • Consider testing multiple antibody concentrations simultaneously

• Cross-reactivity issues:

  • Phospho-specific antibodies may detect related phosphorylated proteins

  • Solution: Validate specificity using dot blot analysis with phosphorylated and non-phosphorylated peptides

  • Include appropriate controls (e.g., MYL9 knockdown samples)

• Incomplete blocking:

  • Insufficient blocking of non-specific binding sites

  • Solution: Extended blocking times (1-2 hours at room temperature)

  • Use of commercial blocking reagents specifically designed for biotin-streptavidin systems

How can I optimize antigen retrieval protocols for different tissue fixation methods when using biotin-conjugated MYL9 antibodies?

Optimizing antigen retrieval for biotin-conjugated MYL9 antibodies requires fixation-specific approaches:

• Formalin-fixed, paraffin-embedded (FFPE) tissues:

  • Heat-induced epitope retrieval (HIER) methods:

    • Citrate buffer (pH 6.0): Standard starting point for MYL9 detection

    • EDTA buffer (pH 8.0-9.0): May improve detection of certain epitopes

    • Optimal conditions: Typically 95-98°C for 20-30 minutes

  • Validated example: MYL9 antibody at 2.5μg/ml successfully stains FFPE human colon smooth muscle tissues

• Fresh-frozen tissues:

  • Minimal retrieval typically required

  • Brief fixation (4% paraformaldehyde for 10-15 minutes) may improve antibody binding

  • Acetone fixation (10 minutes at -20°C) can preserve phospho-epitopes

• Cell preparations:

  • Methanol fixation (-20°C for 10 minutes): Preserves structure while permeabilizing

  • Paraformaldehyde (4%) followed by Triton X-100 (0.1-0.5%) permeabilization

  • Optimize fixation time to balance epitope preservation and accessibility

• Specialized considerations for phospho-epitopes:

  • Include phosphatase inhibitors in all buffers

  • Shorter fixation times often preserve phospho-epitopes better

  • Consider dual retrieval approaches (heat followed by enzymatic digestion)

  • Test multiple retrieval conditions with appropriate positive controls

What strategies can resolve inconsistent results when detecting phosphorylated versus non-phosphorylated forms of MYL9?

Resolving inconsistencies between phosphorylated and non-phosphorylated MYL9 detection requires systematic approach:

• Sample preparation optimization:

  • Rapid sample collection and processing to preserve phosphorylation state

  • Addition of phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Standardized cell lysis conditions to minimize variability

  • Use positive controls like calyculin-treated samples (100 ng/ml for 15-30 minutes)

• Antibody selection and validation:

  • Verify antibody specificity using:

    • Dot blot analysis with phosphorylated and non-phosphorylated peptides

    • Western blotting with phosphatase-treated versus untreated samples

    • Side-by-side comparison with multiple phospho-specific antibodies

  • Consider using highly specific antibodies like Anti-MYL9 (pS20) + MYL12A (pS19) + MYL12B (pS20)

• Technical protocol refinements:

  • For Western blotting:

    • Use freshly prepared reducing agents

    • Optimize transfer conditions (wet transfer often better for small proteins like MYL9)

    • Consider specialized membranes designed for phosphoprotein detection

  • For IHC/IF:

    • Shorter fixation times (10-15 minutes) better preserve phospho-epitopes

    • Use tyramide signal amplification for low-abundance phosphoproteins

• Comparative analysis approach:

  • Run parallel detection of total and phosphorylated MYL9

  • Calculate phosphorylation ratios rather than absolute values

  • Implement internal loading controls for normalization

  • Consider using specialized phosphoprotein staining methods

How can biotin-conjugated MYL9 antibodies be integrated into proximity labeling experiments to identify novel MYL9 interaction partners?

Integrating biotin-conjugated MYL9 antibodies into proximity labeling involves multiple sophisticated approaches:

• BioID-based proximity labeling:

  • Generate MYL9-BioID fusion constructs to biotinylate proteins in close proximity

  • Use biotin-conjugated anti-MYL9 antibodies as controls to validate specificity

  • Combine with mass spectrometry for identification of labeled proteins

  • Recent advances using anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex mixtures, increasing identification of biotinylation sites more than 30-fold compared to streptavidin-based approaches

• APEX2 peroxidase-based proximity labeling:

  • Fuse APEX2 to MYL9 for rapid biotinylation of nearby proteins

  • Live-cell proximity labeling followed by anti-biotin enrichment and mass spectrometry can yield over 1,600 biotinylation sites on hundreds of proteins

  • Compare interactomes from different cellular conditions (e.g., with/without phosphorylation stimulation)

• Crosslinking immunoprecipitation (CLIP) approaches:

  • Use biotin-conjugated MYL9 antibodies for immunoprecipitation

  • Combine with chemical crosslinking to capture transient interactions

  • Particularly useful for studying MYL9's role in actomyosin assembly

• Spatial mapping of interactions:

  • Combine proximity labeling with subcellular fractionation

  • Map interaction networks in different cellular compartments

  • Particularly relevant given MYL9's diverse functions in cytoplasm and potential nuclear roles

What experimental design would best elucidate the relationship between MYL9 phosphorylation and aerobic glycolysis in cancer progression?

Investigating MYL9 phosphorylation and aerobic glycolysis requires a multi-faceted experimental design:

• Comparative phosphoproteomics analysis:

  • Compare phosphorylation patterns between normal and cancer cells using phospho-specific MYL9 antibodies

  • Correlate with expression of glycolytic enzymes (GLUT1, HK2, LDHA)

  • Recent research has shown that MYL9 knockdown significantly inhibits these glycolytic markers

• Functional metabolic studies:

  • Measure lactate production in cells with manipulated MYL9 expression/phosphorylation

  • Data from recent studies show lactate levels are significantly lower in MYL9 knockdown groups

  • Use Seahorse XF analyzer to measure glycolytic rate and mitochondrial function

  • Compare metabolic profiles before and after manipulation of MYL9 phosphorylation

• Signaling pathway analysis:

  • Investigate JAK2/STAT3 pathway activation using phospho-specific antibodies

  • Research indicates MYL9 induces JAK2/STAT3 pathway activity and promotes cancer migration and invasion by enhancing aerobic glycolysis

  • Use pathway inhibitors to establish causality between MYL9, JAK2/STAT3, and glycolysis

• In vivo validation:

  • Develop xenograft models with manipulation of MYL9 phosphorylation status

  • Analyze tumor growth, metastasis, and metabolic profiles

  • Correlate tumor aggressiveness with phospho-MYL9 levels and glycolytic markers

  • Employ PET imaging to assess glucose uptake in tumors

How can biotin-conjugated MYL9 antibodies be used to investigate the potential nuclear roles of MYL9 beyond its cytoplasmic functions?

Investigating nuclear roles of MYL9 using biotin-conjugated antibodies requires specialized approaches:

• Subcellular fractionation and detection:

  • Employ standardized nuclear/cytoplasmic fractionation protocols

  • Use biotin-conjugated MYL9 antibodies for detection in different fractions

  • Include phospho-specific antibodies to determine if phosphorylation status affects localization

  • Validate with confocal microscopy using biotin-streptavidin detection systems

• Chromatin immunoprecipitation (ChIP) analysis:

  • Use biotin-conjugated MYL9 antibodies for ChIP experiments

  • Recent research indicates MYL9 plays a unique role in the nucleus, transcriptionally activating intercellular adhesion molecule 1 (ICAM1)

  • Follow with sequencing (ChIP-seq) to identify genome-wide binding sites

  • Validate findings with reporter gene assays for identified targets

• Proximity-based methods for nuclear interactions:

  • Employ PLA (Proximity Ligation Assay) using biotin-conjugated MYL9 antibodies

  • Target known nuclear proteins (transcription factors, chromatin modifiers)

  • Combine with fluorescence microscopy to visualize nuclear interaction sites

  • Extend to super-resolution microscopy for detailed spatial mapping

• Comparative proteomics of nuclear vs. cytoplasmic MYL9:

  • Immunoprecipitate MYL9 from nuclear and cytoplasmic fractions

  • Identify differential post-translational modifications and binding partners

  • Focus on modifications that might regulate nuclear localization signals

  • Correlate with functional outcomes in gene expression studies